Sex-specific hippocampal metabolic signatures at the onset of systemic inflammation with lipopolysaccharide in the APPswe/PS1dE9 mouse model of Alzheimer’s disease

Highlights • Hippocampal metabolic profile of females is more pro-inflammatory and pro-oxidant.• Comparable LPS-induced sickness behaviour in male and female WT and APP/PS1 mice.• Pro- and anti-inflammatory pathways both recruited 4 h after systemic LPS.• Predominant anti-inflammatory metabolic response to LPS in female hippocampi.


Lack of major metabolic perturbations in the hippocampus of 4.5-month-old APP/PS1 mice
5 of the 98 selected metabolites were found to significantly discriminate between PBS-treated WT and APP/PS1 mice (Table1), albeit predominantly in females. Levels of L-beta-aspartyl-L-glutamic acid, which belongs to the family of N-acyl-alpha amino acids and derivatives which are known for their antiinflammatory action (1), were particularly reduced in female APP/PS1 mice (Suppl. Fig. 2A), but its function and implication in AD pathology is, to the best of our knowledge, unknown. 1-Methyladenosine, an oxidized nucleoside known to be immunosuppressive on macrophage function (2) and found in elevated levels in the urine of patients with mild-to-moderate AD (3), was more abundant in the hippocampus of female APP/PS1 mice compared to their WT female littermates (Suppl. Fig. 2B).
Significant Genotype X Treatment interactions were also found for N-acetyl-(L)-arginine (F(1,34)=12.07, p=0.001), whose levels were significantly lower in PBS-treated APP/PS1 females compared to PBS-treated WT females (Suppl. Fig. 2C), and for the hydrophobic tetrapeptide Asp-Phe-Thr-Thr (F(1,34)=5.40, p=0.03), whose levels were significantly increased in PBS-treated APP/PS1 males compared to their PBS-treated counterparts (Suppl. Fig. 2D). Their function and potential roles in AD pathology are also, to the best of our knowledge, unknown.

Sex differences in the hippocampal metabolic profile are independent of the APP/PS1 genotype.
Forty-one metabolites with sex differences were identified revealing major changes in amino acids, carbohydrate metabolism and fatty acyls (Table 1). A few metabolites from other chemical classes and many unknown metabolites were also found in different levels between PBS-treated males and females (Table 1). Metabolic differences in the methionine and pyruvate metabolic pathways are described in the main manuscript and illustrated Fig. 5 and 6, respectively. Changes in other metabolites with previously associated with differences immune function are described below. Their potential role in brain function or implication in AD progression is presented in Suppl. Table 3.
These included reduced levels of (3R)-beta leucine (Suppl. Fig. 3), a degradation product of the anti-inflammatory amino acid L-Leucine (4), D-alanyl-D-alanine (Suppl. Fig. 3B), an anti-inflammatory antibiotic-binding protein (5), and N(pi)-methyl-L-histidine (Suppl. Fig. 3C), a metabolic product of the amino acid histidine known to be negatively associated with inflammation in obese women (6). Females also presented with an increased abundance of N-Succinyl-L-glutamate 5-semialdehyde (Suppl. Fig. 3D), a metabolite found to be elevated in the plasma of lung cancer patients harbouring a mutation in the epidermal growth factor receptor (7) that also exacerbate their pro-inflammatory status (8).
Differences in amino acid metabolism also indicative of anti-inflammatory effects in females included increased abundance of pantothenate (vitamin B5; Suppl. Fig. 3I), whose dietary intake was found to alleviate chronic low grade inflammation (11) and norepinephrinesulfate (Suppl. Fig. 3O), a metabolite of the anti-inflammatory neurotransmitter norepinephrine (12), as well as reduced levels of (Z)-4-Hydroxyphenylacetaldehyde-oxime (Suppl. Fig. 3J), an enzyme involved in tyrosine metabolism found in increased levels in inflammatory bowel disease (13), and homoarginine (Suppl. Fig. 3K), known to be negatively associated with pro-inflammatory changes (14).
Changes in carbohydrate metabolism and fatty acyls seen in females were indicative of a proinflammatory status. Furthermore, isocitrate, a substrate of the tricarboxylic acid (TCA) cycle found to exert anti-inflammatory effects in a rat model of mild anemia of inflammation (15) and itaconate, a potent anti-inflammatory TCA derivative found in immune cells (16), were also less abundant in the female hippocampus (Suppl. Fig. 3M&N, respectively). Hippocampal concentrations of formyl 3-hydroxybutanoate, a fatty ester, were also significantly lower in females (Suppl. Fig. 3L) and fatty esters are thought to be anti-inflammatory (17).

Metabolites differentially expressed in PBS-and LPS-treated mice regardless of sex and genotype.
Thirty six metabolites were altered to similar extents by LPS in all experimental groups ( Table 1).
The most significant changes were those affecting tryptophan and methionine metabolism, as described in the main manuscript and represented Figs. 4&5, respectively. Changes to other metabolites associated with immune status are described below and represented Suppl. Fig. 4, and the potential association of these metabolites in brain function, sex differences and/or AD progression is described in Suppl. Table 3.
These metabolic differences included increased levels of thymidine and thymine (Suppl. Fig. 4A&B), two derivatives of the anti-inflammatory nucleotide pyrimidine (18) as well as reduced levels of the inflammation signalling molecule adenosine triphosphate (ATP) (19), particularly in WT mice (Suppl. Fig.   4C) and of succinate (Suppl. Fig. 4D), a pro-inflammatory intermediate of the TCA cycle which plays a crucial role in ATP generation (20).

Metabolites showing opposite pattern in LPS-treated males and females
Two of the five metabolites that showed opposite effects of LPS in males and females, S-Adenosy-L-homocysteine Fig. 4E) and N-Succinyl-L-glutamate 5-semialdehyde (Suppl. Fig. 3D), have been reported to be associated with increased inflammation. They both were found more abundant in male hippocampi but less abundant in female hippocampi 4 hours after LPS administration.
Suppl. Table 3. Physiological role of metabolites from known metabolic pathways differently expressed between PBS-treated WT and APP/PS1 mice, PBS-treated males and females and in response to LPS.

Putative metabolite Metabolic Pathway
Physiological role in the brain Implication in sex differences in brain function

Implication in Alzheimer's disease (AD) Implication in immune status
Amino acid metabolism Valine, leucine and isoleucine degradation Degradation product of L-Leucine which is produced by muscle protein catabolism and serves as a donor for brain glutamate synthesis by astrocytes and cerebral protein synthesis (34).

Not known
Increased serum levels of l-leucine in AD patients and in the 3xTg mouse model of AD (35).
L-leucine reduces inflammation and increases repair after muscle injury in rats (4).

Choline
Glycine, serine and threonine metabolism Precursor for the cerebral synthesis of acetylcholine, a neurotransmitter essential for cognitive function, and phospholipid phosphatidylcholine, a major constituent of biological membranes in neurons and glial cells (36,37).
Higher choline concentrations in the hippocampus of cognitively intact elderly females (38).
Loss of cholinergic function in AD is associated with memory decline (39).
Dietary intake of choline improves cognitive function in AD patients and mouse models (37).

L-cystathionine
Glycine, serine and threonine metabolism

Methionine metabolism
Intermediate in the transsulfuration pathway which decreases neurotoxic homocysteine concentrations (40) Mediates the conversion of homocysteine into cysteine (Fig. 5).

Not known
Increased levels in the temporal cortex of post-mortem AD brains (41).
Inhibits the expression of the proinflammatory cytokine MCP-1 in macrophages in vitro (42).

L-methionine Methionine metabolism
Key role in epigenetic regulation in the brain through conversion into homocysteine via S-adenosyl-Lmethionine (43).
No differences in mouse brain concentrations (44).
Decreased levels in the temporal cortex of post-mortem AD brains (41).
Elevated CSF levels in MCI and AD (45).
Excess dietary methionine induces cognitive and neurological hallmarks of AD in mice (46).
Excess dietary methionine induces astrocyte and microglia activation in the hippocampus (46).
No differences in mouse brain concentrations (44).
Increased levels in the postmortem AD brain are associated with cognitive dysfunction and neurological hallmarks of AD (52).
Induces pro-inflammatory activation in endothelial cells in vitro (54).
Not known Not known Not known S-adenosyl-Lmethionine

Methionine metabolism
Arginine and proline metabolism Main donor of methyl groups for DNA methylation in the brain (43).
No differences in mouse brain concentrations (44).
Decreased levels in the postmortem AD brain (56) and CSF of AD patients (57).

5'-methylthioadenosine
Methionine metabolism Arginine and proline metabolism Neuro-protective and antiinflammatory derivative of methionine.
Not known Increased CSF levels in MCI impaired patients (60).

Reduces brain damage; inhibits
INFg and TNFa production and enhances IL-10 production in animal models of neuroinflammation (61 Unclear role in healthy brain function (62).

Not known Not known
Reduced plasma levels associated with increased C-reactive protein levels in chronic kidney disease patients (14).

L-aspartate
Arginine and proline metabolism Accumulation in the brain due to impaired degradation causes brain damage and mental retardation (68).

Not known
Increased circulating levels correlate with inflammation in a subgroup of AD patients (69).
Increased circulating levels in elderly people with chronic low grade inflammation (23).

L-tryptophan
Phenylalanine, tyrosine and tryptophan biosynthesis Tryptophan metabolism Dietary precursor of serotonin and vitamin B3 (nicotinic acid).
Improves mood and cognition by enhancing serotoninergic neurotransmission (70) and nicotinamide pathway (71) Women are more susceptible to episodic memory impairment caused by acute tryptophan depletion (72).
Lower plasma tryptophan levels in females associated with reduced serotonin synthesis rate throughout the brain (73).
Reduced CSF levels in MCI, but not AD, patients (60).
Reduced serotoninergic neurotransmission associated with the development of cognitive symptoms in AD (74).
Upregulation of kynurenine pathway associated with neurological hallmarks of AD (75).
Increases inflammation via stimulation of the kynurenine pathway (76).
See L-tryptophan Elevated CSF levels in MCI and AD (45). See L-tryptophan Pantothenate beta-Alanine metabolism Pantothenate and CoA biosynthesis Vitamin B5. Substrate for the biosynthesis of coenzyme A which contributes to the structure and function of brain cells via its role in the synthesis and oxidation of fatty acids (71).

Not known
Dietary intake positively associated with cerebral Aβ burden in MCI patients (78).
Dietary intake lower systemic inflammation (C-reactive protein levels) in healthy adults over 40 (11).
D-alanyl-D-alanine D-Alanine metabolism Peptidoglycan biosynthesis Not known Not known Not known Anti-inflammatory antibioticbinding protein (5).

Glutathione metabolism
Toxic oxidation product of the antioxidant glutathione produced and exported by astrocytes in the brain (79).
No sex differences in brain tissue content with aging in mice despite the most pronounced decline in glutathione concentrations seen in males (80).
Higher activity of glutathione reductase activity, which catalyses the reduction of GSSG disulphide in glutathione, in the temporal cortex of AD patients (81), but unaltered GSSG contents (82).
Increased circulating levels during acute systemic inflammation in the rats (83).

Methylimidazoleacetic acid
Histidine metabolism Main metabolite of histamine, a neuromodulator, also involved in cognition, wakefulness and anxiety and motivated behaviours (84,85).

Not known
Degeneration of histaminergic nerve fibres in AD (86).
Histamine is produced by immune cells in the brain, induces proinflammatory microglial activation but inhibits LPS-induced microglial activation (87).

N(pi)-methyl-L-histidine Histidine metabolism
Derivative of histidine, a precursor of brain histamine (85). Not known Not known Serum histidine levels are negatively associated with systemic inflammation (C-reactive protein levels) in obese women (6).

Hypotaurine
Taurine and Intermediate in the synthesis of Not known Not known Suppresses inflammatory and hypotaurine metabolism taurine from the methionine derivative cysteine (Fig. 5) in neurons, astrocytes and microglia (88,89).

Succinate
Citrate cycle (TCA cycle) Glyoxylate and dicarboxylate metabolism Support brain energy metabolism by promoting ATP generation in mitochondria (91).

Not known
Reduced whole brain content from 9 months of age in an APP/PS1 mouse model (93).

Isocitrate
Citrate cycle (TCA cycle) Glyoxylate and dicarboxylate metabolism

Not known Not known
Increased CSF levels in a transgenic rat model of tauopathy (95).
Anti-inflammatory in rat model of anaemia of inflammation (15).
Microglial deficiency in isocitrate dehydrogenase, the enzyme that catalyses oxidative decarboxylation of isocitrate, suppresses LP-induced pro-inflammatory cytokine production (TNFa, IL-6, IL-1β) Sex differences in D-lactate metabolism may contribute to reduced association between microbiota and neurological symptoms in females (100).

Methylglyoxal can cause Aβ aggregation (101) and its neurotoxicity is associated with AD (102).
Methylglyoxal is pro-inflammatory and activates glial cells in the brain (103

Glycolysis / Gluconeogenesis
Intermediate metabolite of glucose with potent antioxidant and antiinflammatory properties (106).
Sex differences in pyruvate metabolism associated with reduced oxidative stress and damage in females (107,108). Improves cognitive performance in mouse models of AD without affecting tau or Aβ pathology (109,110).
Ethyl derivatives of pyruvate alleviate pro-inflammatory changes in the brain (111,112 Deficiency in 3PGDH causes brain atrophy, seizures and psychomotor retardation (113).

Not known Not known
3PGDH is an astrocytic enzyme that catalases the production of serine by neurons and glia (114) and is anti-inflammatory in fibroblasts (115

D-sorbitol
Fructose and mannose metabolism Intermediate in the production of fructose from glucose in the brain (120).
Not known Elevated levels in the post-mortem AD brain (121).
Anti-inflammatory properties in resident cells from articular cartilage (122).

Not known
Increased activity of the pentose phosphate pathway associated with increased pro-oxidant activity in the post mortem AD brain (124) Triggers pro-inflammatory cytokines secretion in LPSactivated macrophages (32 Endogenous antibiotic in the brain (125).

Not known Not known
Anti-inflammatory metabolite found in macrophages (16) and produced by microglia (125). Endogenous ligand of benzodiazepine-binding sites in the brain (126).
Not known Increased brain concentration in a transgenic rat model of tauopathy (95).
Reduced brain tissue contents in women (129).
Trends towards reduced levels in the post-mortem AD brain (129).
Suppresses LPS-induced proinflammatory microglial activation in vitro (131). Needed for the synthesis of membranes of neurons and astrocytes during brain development (132).
No sex differences in mouse brain content with normal diet (133).
Reduced levels in the post-mortem AD brain (136).
Accumulates primarily in astrocytes (137) where it triggers the release of TNFα and IL-6 (138).

Biosynthesis of unsaturated fatty acids
Dietary administration, which enters the brain, improves cognitive and motor function (139).
No sex differences in mouse brain content with normal diet (133).
Induces tau phosphorylation and amyloid processing in cultured neurons and astrocytes (134,135,140).
Increased levels in the postmortem AD brain (136).
Impairs the protective migratory and phagocytic activities of microglia in both males and females (141).
Icosatrienoic acid 20:3 Biosynthesis of unsaturated fatty acids Increased brain levels associated with reduced brain growth in developing rats fed with an essential fatty acid deficient diet (142).
Intermediate in the synthesis of Arachidonic acid. Contributes in brain growth and function in combination with other fatty acids (143).
No sex differences in mouse brain content with normal diet (133).
Reduced levels in the post-mortem AD brain (136,144).
Does not trigger pro-inflammatory cytokine release in astrocytes (138).

PGH2-EA Eicosanoids
Metabolite of the endocannabinoid anandamide known to modulate the brain reward system (146) Not known Increases the neurotoxicity of Aβ peptide (147).
Precursor of prostaglandin E2 which mediates LPS-induced sickness (148). Main cellular source of energy in the brain, which can improve cognitive function (150).
Greater ATP production in female mitochondria in the rodent brain (107) Decreased brain contents in a transgenic rat model of tauopathy (95) and mouse model of amyloidosis (151).
Pro-inflammatory signalling molecule in the brain via increased synthesis of prostaglandin E2 (19).